Characterization of three new members of the Arabidopsis thaliana calmodulin gene family: conserved and highly diverged members of the gene family functionally complement a yeast calmodulin null

Abstract.

Three genes encoding members of the EF-hand family of Ca²⁺-binding proteins were identified from Arabidopsis thaliana (L.) Heynh. sequences deposited in the expressed sequence tag and genomic sequence databases. Full-length cDNAs for each of the genes, Cam7, Cam8, and Cam9, were sequenced. Cam7 encodes a conventional 16.8-kDa, 148-amino-acid calmodulin protein (CaM). In contrast, Cam8 and 9 encode highly diverged isoforms of the protein that share 73 and 49% amino acid sequence identity, respectively, with CaM7. RNA gel blot and reverse transcription-polymerase chain reaction experiments revealed that each of the genes is expressed in leaves, flowers and siliques. To test the functional properties of the polypeptides encoded by these genes, they were expressed in Escherichia coli and the yeast Saccharomyces cerevisiae. Each was purified by Ca²⁺-dependent hydrophobic affinity chromatography. CaM7, but neither CaM8 nor CaM9, formed a complex with a basic amphiphilic helical peptide in the presence of Ca²⁺ that could be identified by gel electrophoresis. In spite of these in vitro differences, each of the sequences functionally substituted for yeast CMD1 to maintain viability. Isolation of yeast strains complemented by Cam9 required selection against the plasmid harboring wild-type yeast sequences, whereas complementation by Cam7 and Cam8 did not. These results suggest that the mechanism of action of CaM8 and CaM9 is similar to that of more conventional CaM sequences. CaM9, and to a lesser degree CaM8, however, appear to represent Ca²⁺-binding sensor proteins that interact with a more limited set of target proteins than do more conventional CaM isoforms.